Human growth hormone aqueous formulation

ABSTRACT

A stable pharmaceutically acceptable aqueous formulation containing human growth hormone, a buffer, a non-ionic surfactant, and, optionally, a neutral salt, mannitol, or, a preservative, is disclosed. Also disclosed are associated means and methods for preparing, storing, and using such formulations.

FIELD OF THE INVENTION

The present invention is directed to pharmaceutical formulationscontaining human growth hormone (hGH) and to methods for making andusing such formulations. More particularly, this invention relates tosuch pharmaceutical formulations having increased stability in aqueousformulation.

BACKGROUND OF THE INVENTION

Human growth hormone formulations known in the art are all lyophilizedpreparations requiring reconstitution. Per vial, Protropin® hGH consistsof 5 mg hGH, 40 mg mannitol, 0.1 mg monobasic sodium phosphate, 1.6 mgdibasic sodium phosphate, reconstituted to pH 7.8 (Physician's DeskReference, Medical Economics Co., Orawell, N.J., p. 1049, 1992). Pervial, Humatrope® hGH consists of 5 mg hGH, 25 mg mannitol, 5 mg glycine,1.13 mg dibasic sodium phosphate, reconstituted to pH 7.5 (Physician'sDesk Reference, p. 1266, 1992).

For a general review for growth hormone formulations, see Pearlman etal., Current Communications in Molecular Biology, eds. D. Marshak and D.Liu, pp. 23-30, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989. Other publications of interest regarding stabilization ofproteins are as follows.

U.S. Pat. No. 4,297,344 discloses stabilization of coagulation factorsII and VIII, antithrombin III, and plasminogen against heat by addingselected amino acids such as glycine, alanine, hydroxyproline,glutamine, and aminobutyric acid, and a carbohydrate such as amonosaccharide, an oligosaccharide, or a sugar alcohol.

U.S. Pat. No. 4,783,441 discloses a method for the prevention ofdenaturation of proteins such as insulin in aqueous solution atinterfaces by the addition of up to 500 ppm surface-active substancescomprising a chain of alternating, weakly hydrophilic and weaklyhydrophobic zones at pH 6.8-8.0.

U.S. Pat. No. 4,812,557 discloses a method of stabilization ofinterleukin-2 using human serum albumin.

European Patent Application Publication No. 0 303 746 disclosesstabilization of growth promoting hormones with polyols consisting ofnon-reducing sugars, sugar alcohols, sugar acids, pentaerythritol,lactose, water-soluble dextrans, and Ficoll, amino acids, polymers ofamino acids having a charged side group at physiological pH, and cholinesalts.

European Patent Application Publication No. 0 211 601 discloses thestabilization of growth promoting hormones in a gel matrix formed by ablock copolymer containing polyoxyethylene-polyoxypropylene units andhaving an average molecular weight of about 1,100 to about 40,000.

European Patent Application Publication No. 0 193 917 discloses abiologically active composition for slow release characterized by awater solution of a complex between a protein and a carbohydrate.

Australian Patent Application No. AU-A-30771/89 discloses stabilizationof growth hormone using glycine and mannitol.

U.S. Pat. No. 5,096,885 (which is not prior art) discloses a formulationof hGH for lyophilization containing glycine, mannitol, a non-ionicsurfactant, and a buffer. The instant invention provides an unexpectedlystabilized aqueous formulation in the absence of glycine.

hGH undergoes several degradative pathways, especially deamidation,aggregation, clipping of the peptide backbone, and oxidation ofmethionine residues. Many of these reactions can be slowed significantlyby removal of water from the protein. However, the development of anaqueous formulation for hGH has the advantages of eliminatingreconstitution errors, thereby increasing dosing accuracy, as well assimplifying the use of the product clinically, thereby increasingpatient compliance. Thus, it is an objective of this invention toprovide an aqueous hGH formulation which provides acceptable control ofdegradation products, is stable to vigorous agitation (which inducesaggregation), and is resistant to microbial contamination (which allowsmultiple use packaging).

SUMMARY OF THE INVENTION

One aspect of the invention is a stable, pharmaceutically acceptable,aqueous formulation of human growth hormone comprising human growthhormone, a buffer, a non-ionic surfactant, and optionally, a neutralsalt, mannitol, and a preservative.

A further aspect of the invention is a method of preventing denaturationof human growth hormone aqueous formulations comprising mixing humangrowth hormone and a non-ionic surfactant in the range of 0.1-5% (w/v)(weight/volume). In yet another aspect of the invention, this stabilizedformulation is stored for 6-18 months at 2-8° C.

DESCRIPTION OF THE FIGURES

FIG. 1 is a size exclusion chromatogram of aqueous growth hormoneformulation stored for 28 days at 40° C. (i.e., thermally stressed) andfor one year at 5° C. (i.e., recommended conditions for storage).

FIG. 2 is a plot of Arrhenius rate analysis of growth hormoneaggregation in aqueous formulation.

FIG. 3 is an anion exchange chromatogram comparing a thermally stressed(40° C.) aqueous formulation hGH sample with an aqueous formulation hGHsample stored under recommended conditions (2-8° C.) for one year.

FIG. 4 is a plot of Arrhenius rate analysis of hGH deamidation inaqueous formulation.

FIG. 5 is a graph of the percentage monomer present in the variousformulations where mannitol has been substituted with a neutral salt.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

The following terms are intended to have the indicated meanings denotedbelow as used in the specification and claims.

The terms “human growth hormone” or “hGH” denote human growth hormoneproduced by methods including natural source extraction andpurification, and by recombinant cell culture systems. Its sequence andcharacteristics are set forth, for example, in Hormone Drugs, Gueriguianet al., U.S.P. Convention, Rockville, Md. (1982). The terms likewisecover biologically active human growth hormone equivalents, e.g.,differing in one or more amino acid(s) in the overall sequence.Furthermore, the terms used in this application are intended to coversubstitution, deletion and insertion amino acid variants of hGH, orposttranslational modifications. Two species of note are the 191 aminoacid native species (somatropin) and the 192 amino acid N-terminalmethionine (met) species (somatrem) commonly obtained recombinantly.

The term “pharmaceutically effective amount” of hGH refers to thatamount that provides therapeutic effect in an administration regimen.The compositions hereof are prepared containing amounts of hGH at leastabout 0.1 mg/ml, upwards of about 10 mg/ml, preferably from about 1mg/ml to about 20 mg/ml, more preferably from about 1 mg/ml to about 5mg/ml. For use of these compositions in administration to human patientssuffering from hypopituitary dwarfism, for example, these compositionscontain from about 0.1 mg/ml to about 10 mg/ml, corresponding to thecurrently contemplated dosage regimen for the intended treatment. Theconcentration range is not critical to the invention, and may be variedby the clinician.

B. General Methods

The instant invention has no requirement for glycine. Glycine is anoptional component of the aqueous formulation, although with lessadvantage in the aqueous formulations hereof compared with thoseformulations that are lyophilized for later reconstitution. Amounts ofglycine will range from 0 mg/ml to about 7 mg/ml.

Non-ionic surfactants include a polysorbate, such as polysorbate 20 or80, etc., and the poloxamers, such as poloxamer 184 or 188, Pluronic®polyols, and other ethylene/polypropylene block polymers, etc. Amountseffective to provide a stable, aqueous formulation will be used, usuallyin the range of from about 0.1% (w/v) to about 5% (w/v), morepreferably, 0.1% (w/v) to about 1% (w/v). The use of non-ionicsurfactants permits the formulation to be exposed to shear and surfacestresses without causing denaturation of the protein. For example, suchsurfactant-containing formulations are employed in aerosol devices suchas those used in pulmonary dosing and needleless jet injector guns.

Buffers include phosphate, Tris, citrate, succinate, acetate, orhistidine buffers. Most advantageously, the buffer is in the range ofabout 2 mM to about 50 mM. The preferred buffer is a sodium citratebuffer.

A preservative is included in the formulation to retard microbial growthand thereby allow “multiple use” packaging of the hGH. Preservativesinclude phenol, benzyl alcohol, meta-cresol, methyl paraben, propylparaben, benzalconium chloride, and benzethonium chloride. The preferredpreservatives include 0.2-0.4% (w/v) phenol and 0.7-1% (w/v) benzylalcohol.

Suitable pH ranges, adjusted with buffer, for aqueous hGH formulationare from about 4 to 8, more preferably about 5.5 to about 7, mostadvantageously 6.0. Preferably, a buffer concentration range is chosento minimize deamidation, aggregation, and precipitation of hGH.

Mannitol may optionally be included in the aqueous hGH formulation. Thepreferred amount of mannitol is about 5 mg/ml to about 50 mg/ml. As analternative to mannitol, other sugars or sugar alcohols are used, suchas lactose, trehalose, stachiose, sorbitol, xylitol, ribitol,myoinositol, galactitol, and the like.

Neutral salts such as sodium chloride or potassium chloride areoptionally used in place of sugars or sugar alcohols. The saltconcentration is adjusted to near . isotonicity, depending on the otheringredients present in the formulation. For example, the concentrationrange of NaCl may be 50-200 mM, depending on the other ingredientspresent.

In a preferred embodiment, the formulation of the subject inventioncomprises the following components at pH 6.0.

Ingredient Quantity (ma) hGH 5 Sodium Chloride 8.8 Polysorbate 20 2.0Sodium citrates 2.5 Phenol 2.5 Sterile water 1 ml

It will be understood that the above quantities are somewhat flexiblewithin ranges, as set forth in more detail above, and that the materialsare interchangeable within the component categories. That is,polysorbate 80, or a poloxamer, may be substituted for polysorbate 20, asuccinate or acetate buffer could instead be employed, and alternativepreservatives and different pHs could be used. In addition, more thanone buffering agent, preservative, sugar, neutral salt, or non-ionicsurfactant may be used. Preferably, the formulation is isotonic andsterile.

In general the formulations of the subject invention may contain othercomponents in amounts not detracting from the preparation of stableforms and in amounts suitable for effective, safe pharmaceuticaladministration. For example, other pharmaceutically acceptableexcipients well known to those skilled in the art may form a part of thesubject compositions. These include, for example, various bulkingagents, additional buffering agents, chelating agents, antioxidants,cosolvents and the like; specific examples of these could includetrimethylamine salts (“Tris buffer”), and disodium edetate.

EXPERIMENTAL EXAMPLES A. Assay Methods

Anion exchange chromatography (HPIEC) was run on a TSK DEAE 5 PW column(1.0×7.5 cm) at 45° C. with a flow rate of 0.5 ml/min. The column wasequilibrated in 50 mM potassium phosphate, pH 5.5, containing 10% (w/v)acetonitrile. Elution was performed using a 25 minute gradient from50-100 mM potassium phosphate, pH 5.5 with constant 10% (w/v)acetonitrile. The column load was 83 μg of protein. Detection was at 230nM.

Nondenaturing size exclusion chromatography was run on a TSK 2000 SWXLcolumn in 50 mM sodium phosphate, pH 7.2 containing 150 mM sodiumchloride. The flow rate was 1 ml/min, with a 50-75 μg column load anddetection at either 214 and 280 nm.

Denaturing size exclusion chromatography was run on a Zorbax GF250column in 200 mM sodium phosphate, pH 6.8-7.2/0.1% SDS. The flow ratewas 1.0 ml/minute, with a with a 50-75 μg column load and detection ateither 214 and 280 nm.

B. Formulation Preparation

In general, aqueous hGH formulation samples for analysis in theseexperimental examples were prepared by buffer exchange on a gelfiltration column. The elution buffer contained either sodium chlorideor mannitol, buffer and the non-ionic surfactant in their final ratios.This resulting solution was diluted to a desired hGH concentration andthe preservative was added. The solution was sterile filtered using asterilized membrane filter (0.2 micron pore size or equivalent) andfilled into sterile 3 cc type 1 glass vials, stoppered and sealed withaqueous-type butyl rubber stoppers and aluminum flip-off type caps.

The aqueous hGH formulation used in the experimental examples consistedof 5.0 mg somatropin (Genentech, Inc.), 45.0 mg mannitol, 2.5 mg phenol,2.0 mg polysorbate 20, and 2.5 mg sodium citrate, pH 6.0, per ml ofsolution. The lyophilized formulation used as a reference for comparisonin the examples consisted of 5.0 mg somatropin, 1.7 mg glycine, 45.0 mgmannitol, 1.7 mg sodium phosphate, 9 mg benzyl alcohol per ml sterilesolution after reconstitution.

C. Example I Chemical Stability of the Aqueous Formulation

Vials of the hGH aqueous formulation (lots 12738/55-102 and12738/55-105) were incubated at either recommended storage temperaturesof 2-8° C., or elevated storage temperatures of 15° C., or 25° C., andthen removed at various time points and assayed for changes in pH, colorand appearance, and protein concentration. In addition, samples wereincubated at 40° C. in order to study degradation patterns under extremestress conditions. Degradation patterns for the aqueous formulation werealso compared to the known degradation patterns for lyophilized growthhormone.

After storage at 2-8° C. for up to one year, the aqueous formulationshowed insignificant changes in pH, color and appearance, and proteinconcentration. Nondenaturing size exclusion HPLC performed on samplesstored for up to one year at 2-8° C. showed no significant aggregationof the drug product (FIG. 1). This result is unexpected in light of theteaching of U.S. Pat. No. 5,096,885 that glycine contributes topreventing aggregation in the lyophilized preparation.

At temperatures above 8° C., little or no changes in pH or proteinconcentration were observed over time. Visual inspection revealed anincrease in opalescence with time for samples stored at 40° C. Thischange was minimal during storage at 15-25° C. and has not been observedduring 2-8° C. storage.

The amount of degradation product was calculated as an area percentageof the total hGH area of the chromatogram. The rate constant for eachreaction was then calculated by subtracting the percentage ofdegradation product from 100%, taking the log10, and plotting againstthe time in days. The slope of a straight line to fit these data wasused as the reaction constant (k). Arrhenius analysis was done byplotting the natural logarithm (ln) of the absolute value of eachcalculated reaction rate constant at 15, 25, and 40° C. as a function ofthe inverse absolute temperature and then extrapolating to 5° C.Arrhenius and real time rate analysis (FIG. 2) of data from the sizeexclusion HPLC indicate that the amount of growth hormone aggregationafter 18 months of storage will be less than 1% (w/v).

Anion exchange HPLC analysis performed on the aqueous hGH formulationstored at 40° C. indicated an increase in acidic peaks over 28 days(FIG. 3). Three of these peaks, eluting at about 16, 17.5, and 26minutes, were produced by hGH deamidation at positions 149, 152, and 149plus 152. Arrhenius and real time rate analysis (FIG. 4) of data fromthis method, were plotted as described above, and indicate that theamount of deamidated hGH in these lots after 18 months of storage at2-8° C. will be about 9% (w/v). This includes an initial amount of about2.4% (w/v) deamidated hGH at time zero. Values as high as 15% (w/v)deamidation have been reported for other hGH products (Larhammar, H., etal., (1985) Int. J. Pharmaceutics 23:13-23). Although the rate ofdeamidation is faster in the aqueous state, this rate is minimized at pH6.0 and below.

D. Example II

Physical Stability of the Aaueous Formulation

Each of six vials of lyophilized growth hormone were reconstituted with1 ml bacteriostatic water for injection (BWFI) U.S.P. After dissolving,the contents were transferred to 3 cc vials, stoppered, and capped toprovide the same configuration as that for the aqueous formulation. Thesix vials of the hGH aqueous formulation and six vials of reconstitutedlyophilized hGH were vigorously shaken top to bottom in a horizontalfashion on a Glas-Col Shaker-in-the-Round at 240 jolts per minute usinga stroke setting of 2.5, giving a horizontal displacement of 8±1 cm forup to 24 hours at room temperature to assess the effects of agitation onphysical stability of the hGH aqueous formulation. All twelve sampleswere placed in a straight line on the shaker to assure that they wereall exposed to the same force for each formulation. Two vials wereremoved for assays at 30 minutes, 6 hours, and 24 hours.

The results are displayed in Table I. Agitation produced very littlechange in the visual clarity of the aqueous formulation. There was nochange in the content of total growth hormone monomer as detected by anondenaturing size exclusion HPLC assay. This assay detects noncovalentaggregates, which are completely dispersed by SDS in a denaturing sizeexclusion HPLC assay.

By comparison, these results also demonstrated that the reconstitutedlyophilized product was more sensitive to treatment, even after only 30minutes of shaking. This sensitivity is typical for all currentlyavailable formulations of hGH, other than the aqueous formulation of theinstant invention. The inclusion of the non-ionic surfactant is the mostimportant factor in preventing this phenomenon from occurring.

TABLE I Effects of Agitation at Room Temperature on hGH AqueousFormulation vs. Reconstituted Lyophilized Formulation % % HPSEC Soluble% Total¹ Sample Color/Appearance Monomer Protein Monomer UnshakenAqueous clear/colorless 99.7 ND ND Aqueous clear/colorless 99.9 ND NDLyophilized clear/colorless 99.0 100 99.0 Lyophilized clear/colorless NDND ND Shaken 0.5 hr Aqueous very slightly 99.9 100 99.9opalescent/colorless Aqueous very slightly 100.0 100 100.0opalescent/colorless Lyophilized slightly opalescent/ 93.6 100 93.6colorless Lyophilized clear/colorless 92.8 100 92.8 Shaken 6 hr Aqueousslightly opalescent/ 99.9 100 99.9 colorless Aqueousopalescent/colorless 99.8 100 99.8 Lyophilized very opalescent/ 80.5 7358.8 yellow to brown Lyophilized very opalescent/ 72.7 61.7 44.9 yellowto brown Shaken 24 hr Aqueous slightly opalescent/ 99.8 100 99.8colorless Aqueous clear/colorless 99.8 ND ND Lyophilized verycloudy/yellow 60.6 21.5 13.0 to brown Lyophilized very cloudy/yellow56.7 14.8 8.4 to brown ¹Total monomer = (% monomer × % solubleprotein)/100

E. Example III Preservative Effectiveness in the Aqueous Formulation

Samples of hGH aqueous formulation were subjected to bacterial challengeaccording to an abbreviated challenge using the standard U.S.P. test. Inthis test, a suspension of either E. coli or S. aureus was added to analiquot of hGH aqueous formulation to give a final concentration ofbacteria between 10⁵ to 10⁶ CFU/ml. Viable bacteria remaining in thetubes were counted immediately and after 4 and 24 hours incubation at20-25° C. The percentage change in the concentration of themicroorganisms during the challenge was calculated according to thefollowing equation:

${\% \mspace{14mu} {initial}\mspace{14mu} {titer}} = \frac{{{titer}\mspace{14mu} {at}\mspace{14mu} T} = {X\mspace{14mu} {hours} \times 100}}{{{titer}\mspace{14mu} {at}\mspace{14mu} T} = 0}$

The results of this experiment indicated that for two species ofbacteria, concentrations of viable bacteria were reduced to less than0.01% of the initial concentrations after 24 hours.

F. Example IV Substitution of Mannitol with Salt

In this experiment aqueous formulations of hGH were compared that variedin concentrations of salt, mannitol, and non-ionic surfactant. Allformulations contained 5 mg/ml hGH/ 0.25% (w/v) phenol/10 mM sodiumcitrate, pH 6.0. Samples were stored 3-4 months at 2-8° C. FIG. 5indicates the percentage monomer present in the indicated formulations.The Table below indicates the composition of each formulation. Theseresults demonstrate the unexpected stability of hGH in a formulation inwhich mannitol has been substituted with a neutral salt in the presenceof a surfactant.

TABLE 3 Formulations Tested in FIG. 5 Formulation # Composition 42 0.1%(w/v) polysorbate 20 50 mM mannitol 47 0.1% (w/v) poloxamer/188 0.1MNaCl 51 0.5% (w/v) polysorbate 20 50 mM mannitol 52 0.1% (w/v) poloxamer188 50 mM mannitol 53 0.1% (w/v) poloxamer 184 50 mM mannitol 60 0.2%(w/v) polysorbate 20 0.1M NaCl 61 0.2% (w/v) polysorbate 20 0.05M NaCl62 0.2% (w/v) polysorbate 20 0.15M NaCl 63 0.2% (w/v) polysorbate 20 50mM mannitol

1-21. (canceled)
 22. An aqueous formulation of human growth hormonecomprising: a) at least about 0.1 mg/ml of human growth hormone; b) abuffer having a concentration in the range of about 2 mM to about 50 mM;c) a non-ionic surfactant in the range of from a 0.1% (w/v) to about 5%(w/v); d) optionally, mannitol, e) optionally, a neutral salt at aconcentration adjusted to near isotonicity, and f) optionally, apreservative.
 23. The formulation of claim 22 wherein amount of bufferis adjusted to provide a pH of from about 4 to
 8. 24. The formulation ofclaim 22 wherein the non-ionic surfactant is a poloxamer.
 25. Theformulation of claim 22 wherein the non-ionic surfactant is polysorbate.26. The formulation of claim 24 wherein the poloxamer is poloxamer 188or poloxamer 184 in the range of from a 0.1% (w/v) to about 1% (w/v).27. The formulation of claim 25 wherein the polysorbate is polysorbate20 or polysorbate 80 in the range of from a 0.1% (w/v) to about 1%(w/v).
 28. The formulation of claim 1 wherein the concentration ofmannitol is about 5 mg/ml to about 50 mg/ml.
 29. The formulation ofclaim 23 wherein the buffer is an alkali metal citrate buffer.
 30. Theformulation of claim 23 wherein the buffer is selected from the groupconsisting of phosphate, Tris, succinate, acetate, or histidine buffers.31. The formulation of claim 22 wherein the formulation is sterilefiltered using a 0.2 micron pore size sterilized membrane filter. 32.The formulation of claim 22 wherein the preservative is selected fromphenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben,benzalconium chloride and benzethonium chloride.
 33. The formulation ofclaim 22 wherein the neutral salt is an alkali metal halide.
 34. Theformulation of claim 22 wherein the preservative is phenol in the rangeof 0.2-0.4% (w/v).
 35. The formulation of claim 22 wherein thepreservative is benzyl alcohol in the range of 0.7-1% (w/v).
 36. Theformulation of claim 22, wherein the formulation comprises 5 mg/ml hGH,8.8 mg/ml sodium chloride, 2.0 mg/ml polysorbate 20, 2.5 mg/ml sodiumcitrate, and 2.5 mg/ml phenol, at pH 6.0.
 37. A method of preventingdenaturation of a human growth hormone formulation, the methodcomprising mixing an aqueous solution of human growth hormone and anon-ionic surfactant in the range of 0.1-5% (w/v).
 38. The method ofclaim 37 wherein the non-ionic surfactant is a poloxamer.
 39. The methodof claim 37 wherein the non-ionic surfactant is a polysorbate.
 40. Themethod of claim 38 wherein the poloxamer is poloxamer 188 or
 184. 41.The method of claim 39 wherein the polysorbate is polysorbate 20 orpolysorbate 80.